Project summary Alcohol is linked to approximately half of all violent crimes committed in the United States. Severe social and financial ramifications of these acts of violence include chronic health issues, pain and suffering, property damages, repeated arrests, incarceration, and fatalities; financially, the consequences of alcohol-associated violence will cost the US more than $200 billion this year alone. Advancing our understanding of the biological basis for violent outbursts may guide the development of clinical tools for the diagnosis, treatment and prevention of alcohol-associated aggression. According to a frequently cited, yet presently untested hypothesis, alcohol may disrupt medial prefrontal cortical (mPFC) top-down control over subcortical, aggression-promoting brain regions. On a cellular level, alcohol may hinder excitatory inputs onto inhibitory GABAergic interneurons in the mPFC; reduced GABAergic transmission may subsequently disinhibit excitatory corticolimbic projections. To address this hypothesis, chemogenetic or optogenetic tools will be used to target GABAergic interneurons in the mPFC of male mice; analyses of intruder-directed murine aggression will clarify whether reducing inhibitory interneuron activity in the mPFC is sufficient to escalate aggression. Wild- type (wt) and mutant male mice with Cre recombinase (Cre) specifically expressed in fast-spiking parvalbumin- positive interneurons (PV-Cre mice) will be microinfused with an adeno-associated virus (AAV) to drive Cre- dependent expression of inhibitory designer receptors exclusively activated by designer drugs (DREADDs) in the mPFC. After viral infection, PV-Cre mice will be characterized as alcohol-heightened (AHA) or alcohol non- heightened aggressors (ANA) and tested for escalated aggression toward a submissive conspecific upon inhibition of PV+ interneurons with doses of clozapine-N-oxide. The second aim addresses the role of alcohol in the disinhibition of aggression; associated experimental work will test whether alcohol-heightened aggression can be blocked via activation of inhibitory PV+ interneurons in the mPFC. PV-Cre mice and wt controls will be microinfused into the mPFC with an AAV driving Cre-dependent expression of stable stepwise function opsins (SSFOs) in PV+ interneurons. To assess the effects of PVI activation on alcohol- heightened aggression, characterized mice will receive a pulse of blue light for stable depolarization of PVIs via activated SSFOs prior to receiving water or a pro-aggressive dose of alcohol. In sum, experiments guided by aims one and two will test the following hypotheses: 1.) Inhibition of PV+ interneurons in the mPFC is sufficient to escalate aggression and, 2.) Reduced activity by mPFC inhibitory interneurons is required for expression of alcohol-heightened aggression. This work will provide novel insight into the involvement of mPFC microcircuitry in alcohol-heightened aggression and will encourage further investigation into the neurochemical and genetic bases for alcohol-related violence in humans.